Diabetes is an epidemic in USA and Veterans are nearly three times as likely as the general population to have diabetes. Patients with diabetes have an increased risk of developing cardiovascular disease and more than 75 percent of them die of cardiovascular complications. Given that the prevalence of type 2 diabetes continues to increase steadily and the prevalence of CVD in patients with type 2 diabetes continues despite advances in the management of CVD, it is crucial to develop new therapeutic strategies for cardiovascular disease to reduce mortality in diabetic patients. In recent years, a large number of studies have provided strong evidence that lipopolysaccharide (LPS)-activated toll-like receptor (TLR)4 plays an important role in chronic inflammation and atherosclerosis in diabetes. Since type 2 diabetes is also associated with an increased saturated fatty acids (SFAs), we have investigated the impact of SFAs on LPS stimulated TLR4 activation in macrophages. Interestingly, we found that palmitic acid (PA), the most abundant SFAs in diabetes, amplified LPS-triggered TLR4 signaling on inflammatory gene expression in macrophages by 4-fold. We also found that acid sphingomyelinase (aSMase) plays a key role in the amplification of TLR4-mediated inflammatory signaling by PA. Overall, our recent studies have elucidated an interaction between TLR4 signaling and sphingolipid metabolic pathway: LPS and PA have a synergy on aSMase-mediated sphingomyelin hydrolysis and subsequent ceramide/sphingosine 1 phosphate (S1P) increase while the ceramide/S1P increase in turn amplifies TLR4-mediated signaling. Since all our above findings were made in vitro, it is important to confirm the observations in vivo. Thus, we proposed to determine if high-fat diet (HFD) enriched with PA and LPS administration increase atherosclerosis cooperatively via sphingolipids. We proposed animal studies to confirm our in vitro observations and studies with macrophages to explore the mechanisms involved in the synergy between PA and TLR4 signaling. Specific objective 1: To determine if HFD enriched with PA enhances the stimulatory effect of LPS on atherosclerosis in diabetic LDL receptor-deficient (LDLR-/-) mice and if aSMase plays an essential role in the enhancement. We hypothesized that HFD enriched with PA enhances LPS-induced atherosclerosis in diabetic LDLR-/- mice, and aSMase-mediated sphingolipid metabolism plays an essential role in the enhancement. Specific objective 2: To determine the mechanisms involved in the synergy of PA and LPS on S1P production and the role of S1P in IL-6 upregulation. We hypothesized that LPS and PA exert a cooperative stimulation on sphingosine kinase (SK), and S1P augments the inflammatory signal stimulated by LPS and PA by enhancing NF?B signaling. Specific objective 3: To determine the mechanisms involved in the cooperative activation of aSMase by LPS and PA. We hypothesized that LPS and PA have a cooperative stimulation on protein kinase C (PKC)?nd aSMase membrane translocation, leading to increased aSMase activity and ceramide production. METHODS: For Objective 1, systemic inflammation will be induced by administration of low dose LPS, and HFD enriched with PA will be given to LDLR-/- mice to induce diabetes. We will then determine sphingolipids, vascular inflammation and atherosclerosis using lipidomics, histology, and immunohistochemistry. We will also employ LDLR-/- mice with aSMase-deficiency to determine the role of aSMase in diabetes-accelerated atherosclerosis. For Objective 2, we will perform cell and molecular biology studies. This study will provide novel insight into the interaction between inflammation and dyslipidemia in diabetes-accelerated atherosclerosis and is important for developing new therapeutic strategies. The incidence of diabetes in Veterans is much higher than that in general population. This research project will provide important information for developing new therapeutic strategies to prevent fatal cardiovascular events in our Veterans with diabetes.